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  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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  • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
  • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
  • C12Y207/01074—Deoxycytidine kinase (2.7.1.74)
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
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  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16061—Methods of inactivation or attenuation
  • C12N2740/16062—Methods of inactivation or attenuation by genetic engineering

Definitions

• the present invention concerns a new method of directing evolution of a target polynucleotide of interest (genomic region, gene, coding sequence or any polynucleotide) for obtaining variants of this target polynucleotide that confer a desired phenotype to target cells, especially to mammalian cells, in particular human cells.
• the present invention concerns a method to generate genetic variability by preparing a cell library as well as a method to isolate variants of a polynucleotide or variants of a protein able to impact the phenotype of a cell, and to identify theses polynucleotide variants or protein variants responsible for this phenotype.
• Randomised mutagenesis of genes of interest has recently deserved increased attention.
• a library of mutated genes is first generated and then screened for the presence of mutants possessing a given property either in vitro or, if possible, through genetic screening in bacterial cells.
• mutants possessing a given property either in vitro or, if possible, through genetic screening in bacterial cells.
• the properties observed in vitro for a given mutant often do not result in the desired phenotype when introduced in the eukaryotic cell.
• mutants isolated for their ability to carry out a specific enzymatic activity as the phosphorylation of thymidine residues or thymidine kinase (TK) activity in vitro or in bacteria, proved to be inefficient to confer a TK + phenotype to TK-human cells in culture.
• Tet system tetracycline-regulated gene expression system
• Das AT., Zhou X., Vink M., Klaver B., Verhoef K., et al. (2004) J. Biol. Chem., 279: 18776-82 In Das et al., replication-competent viruses (functional viruses) were constructed, and genomic expression was driven by the Tet system. By selecting for the faster replicating variants, an improved Tet system was selected.
• selection was necessarily coupled to viral replication capacity, and its applications extremely limited.
• HIV human immunodeficiency virus
• Another approach is to generate a bank of mutated genes by degenerated polymerase chain reaction (PCR), followed by insertion of the thereby generated library in bacteria.
• PCR polymerase chain reaction
• a common step after the generation of the library is then the screening for the mutant possessing the desired phenotype, generally in vitro or, when possible, in bacteria having the appropriate genetic background for such screening.
• these pre-screening tests often lead to the isolation of mutants that actually do not confer the desired phenotype to the human cell.
• Direct screening of the library in the human cell by inserting the genes by transfection is also made difficult by the low efficiency of generation of stable clones by this method, which dramatically reduces the complexity of the library, and by the problem that this procedure generates, of the insertion of multiple copies of the gene in the cell genome. This hampers clonal screening.
• Insertion of the library in lentiviral vectors allows clonal screening quite easily, instead.
• transposing the library from the bacteria to the lentiviral vectors also suffers from a drastic low efficiency that reduces the complexity of the library. Generating the library directly in these vectors, instead, bypasses this problem.
• Figure 1 is a schematic diagram outlining the advantages of the present invention over preexisting methods to generate diversity in cells (for example mammalian or human cells).
• Figure 2 is a schematic diagram of the different steps of the method of the present invention.
• Figure 3 is schematic diagram of the general structure of the genomic RNAs (genetic material) used in the method of the present invention.
• the first schematic diagram represents a genetic material with a promoter and the target polynucleotide (A).
• the second schematic diagram represents a genetic material with a first promoter driving the expression of the target polynucleotide, and a selectable marker gene (marker) under the control of a second promoter (B).
• the third schematic diagram represents a preferred embodiment of the genetic material, wherein the first promoter is EF1 oc, the second promoter is human (h) PGK and the selectable marker gene is the puromycin gene.
• ⁇ psi sequence
• RRE Rev responsive element
• PPT polypurine tract
• FLAP cPPT-CTS triplex region (C).
• the last schematic diagram represents the general structure of another preferred embodiment of genetic material; arrows noted “EF1 -CR” and “PGK-CR” are primers designed to amplify the sequence of the target polynucleotide (D)
• Figure 4 is a schematic diagram illustrating the procedure followed to produce two subsequent viral generations (Fn and Fn+1 ) (A).
• a selection step is applied, herein with treatment with puromycin (though any selectable marker may be used) (B).
• B The optional selection of target cells possessing the desired phenotype is also indicated.
• FIG. 5 is a schematic diagram illustrating the overtransduction step that can be implemented within the method of the present invention.
• the below schematic diagram represent the overtransduction step, and shows two overtransductions (A) in the general procedure and (B) in an specific example where the overtransduction step is used to implement at the 6 th (F6) generation of the evolution of the dCK gene.
• Figure 6 is a diagram illustrating (A) sixteen (16) independent variants of the dCK target polynucleotide obtained at the F8 generation, and (B) 10 clones obtained at the F16 generation (B) using the method of the invention. At the top, the 14 positions that have been found modified in at least one of the 16 clones analysed are indicated by bars and their nucleotide position is given.
• Figure 7 is a diagram illustrating three (3) types of selection steps conducted within the method of the present invention: (i) positive selection on a negative genetic background, (ii) negative selection on a negative genetic background, and (iii) positive selection for a secreted protein.
• Figure 8 discloses the cell survival with regard to increasing concentrations of AraC ( ⁇ ) in presence of the wild type dCK gene (white triangles and solid thick curve) or variants containing point mutations or deletion in the dCK gene (other curves). Each plot represents the average of three independent experiments. Thin solid lines and grey circles: variants giving a death rate comparable to wt dCK one; dotted thin line and white circles: variant giving a death rate lower than the wt dCK one; Dotted thick line and black squares: variant giving a death rate higher than the dCK one.
• Figure 9 is a diagram illustrating the screening methodology implemented to identify Messa 10K cells transduced with dCK variants more sensitive to Gemcitabine than wild type Messa 10K cells.
• Figure 10 discloses (A) the cell survival with regard to increasing concentrations of Gemcitabine (nM) in untransduced Messa 10K cells (grey solid line), in presence of the wild type dCK gene (large dashed lines) or in presence of dCK G12 variants gene (black solid lines); (B) the survival of Messa 10K cells in presence of 75 nM Gemcitabine in untransduced Messa 10K cells (Messal OK), in Messa 10K cells transduced with a viral vector carrying a wild type dCK gene (M-dCK), in Messa 10K cells transduced with a viral vector carrying the E8 variant dCK gene with mutations G>A 739 et OA 745 (M-E8) or in Messa 10K cells transduced with a viral vector carrying the G12 variant dCK gene (M-G12), and (C) Western Blot to detect dCK expression on cell lysates, using a polyclonal rabbit antibody; M: untransduced Messal OK
• Gemcitabine nM
• Figure 12 is a diagram representing the LTR region of a lentiviral vector obtained at the 8 th generation, and showing the mutations obtained in the U3 region of these evolved vectors.
• Figure 13 represents (A) the sequence of the wild type LTR of HIV-1 (SEQ ID NO:5) and (B) the sequence of the mutated LTR as defined in SEQ I D NO:6. Mutations are underlined, and numbered according to the +1 nucleotide of SEQ I D NO:5.
• the present application describes a method, which is based on the insertion of at least one target polynucleotide in retroviral-derived vectors, and on the abilities of the viral replication machinery to drive the evolution of these at least one target polynucleotide and to generate a library of variants directly in the appropriate vector, for delivery to target cells.
• the present invention relates to a cell culture (in vitro) method for generating genetic diversity in the nucleotide sequence of a target polynucleotide, comprising:
• step b) transducing producer cells with the parental replication-defective lentiviral particles of step a), optionally in the presence of a mutagen, to obtain parental cells;
• step b) transcomplementing the parental producer cells of step b) with viral proteins necessary for the production of a first filial generation of replication-defective lentiviral particles (F1 );
• e repeating, n consecutive times, with n being 1 or more, the at least three following steps: e1 ) transducing fresh producer cells with the immediately previous filial generation of replication-defective lentiviral particles, optionally in the presence of a mutagen, thereby obtaining a Fn filial cell generation;
• step e2 transcomplementing the Fn producer cells of step e1 ) with viral proteins necessary for the production of a n+1 generation of replication-defective lentiviral particles (Fn+1 ); and e3) collecting the n+1 generation of replication-defective lentiviral particles (Fn+1 );
• replication-defective lentiviral particles contain the target polynucleotide or a nucleotide variant of the target polynucleotide.
• the first step in the method is to provide parental replication-defective lentiviral particles which comprise the genetic material necessary for the remaining of the method.
• a lentiviral particle is constituted of both proteins and a genetic material that is encapsidated into these proteins. Particles are made of viral envelope proteins (encoded by an env gene) as well as structural proteins (encoded by the gag gene). Inside the particle, a viral core (or capsid) is found, formed of three enzymes (encoded by the pol gene) i.e., the reverse transcriptase, the integrase and the protease, and of genetic material.
• the genetic material is present in the particle under the form of two molecules of RNA.
• Each molecule of RNA which can be the same or different, comprises regulatory elements necessary for the transfer, transcription and optionally expression of the genetic material into the genome of a cell host and at least one target polynucleotide.
• the regulatory elements necessary for the transfer, transcription and optionally expression of the genetic material in the genome of a cell host are well known in the art and comprises LTR (both in 5' and 3'), the encapsidation sequence (named psi or ⁇ ) which is a sequence necessary for the encapsidation of the RNA molecules when the particle is formed, the primer-binding site (PBS), the PPT (polypurine tract) sequence, and the RRE (Rev-responsive element), a sequence required for the binding of viral Rev protein and efficient packaging of the genetic material in the particles.
• LTR both in 5' and 3'
• the encapsidation sequence named psi or ⁇
• PBS primer-binding site
• PPT polypurine tract
• RRE Rev-
• LTR long-terminal repeat
• retroviral DNA DNA sequence found in retroviral (preferably lentiviral) DNA at both ends of the reverse transcription product.
• An example of LTR is the one of HIV-1 as defined in SEQ ID NO: 5.
• the region U3 is from nucleotides 1 to 455
• the R region is from nucleotides 456 to 551
• the U5 region is from nucleotides 552 to 635.
• LTRs drive the expression of genomic and subgenomic RNAs, both in retroviruses and in retroviral-derived vectors (preferably lentivirus or lentiviral-derived vectors).
• LTRs are partially transcribed into an RNA intermediate, followed by reverse transcription into complementary DNA (cDNA) and ultimately into dsDNA (double-stranded DNA) with new full LTRs.
• the LTRs also mediate integration of the retroviral DNA via an LTR- specific transposase called integrase into the host cell's chromosomal DNA. As is known in the art, this is the basic mechanism of integration used by the human immunodeficiency virus (HIV).
• HAV human immunodeficiency virus
• 3' LTR refers to a 3' retroviral or lentiviral long terminal repeat, which may or may not be modified from its corresponding native 3' LTR (i.e., existing in the wild type retrovirus) by deleting and/or mutating endogenous sequences and/or adding heterologous sequences.
• 5' LTR refers to a 5' retroviral or lentiviral long terminal repeat, which may or may not be modified from its corresponding native 5' LTR by deleting and/or mutating endogenous sequences and/or adding heterologous sequences. LTRs may be natural or synthetic.
• the genetic material also comprises sequences that have been identified to be involved in the improvement of nuclear import of the reverse transcribed DNA and thus in the improvement of integration into the host cell: cis-acting cPPT and CTS sequences forming the so-called flap sequence (Zennou V. et al. (2000) Cell, 101 , 173-185).
• cis-acting cPPT and CTS sequences forming the so-called flap sequence (Zennou V. et al. (2000) Cell, 101 , 173-185).
• a plasmid containing these cPPT- CTS sequences is particularly useful to transduce non-dividing cells.
• the cPPT and CTS regions are inserted in the vector in order to a form a flap sequence i.e., a sequence able to adopt a triplex structure after retrotranscription.
• sequences originating from retroviruses or lentiviruses are used as a DNA fragment isolated from its natural (viral genome) nucleotide context i.e., out of the context of the gene or region in which it is naturally contained in the retrovirus or lentivirus genome. Therefore, the sequence is used, in the present invention, deleted from the unnecessary 5' and 3' parts of its natural gene or region, and is recombined with sequences of different origin. These sequences may be either prepared synthetically (chemical synthesis) or by amplification of the lentiviral or retroviral DNA, such as by Polymerase chain reaction (PCR).
• PCR Polymerase chain reaction
• lentivirus or “lentiviraf denotes a category of retroviruses particularly preferred for the present invention.
• lentiviruses include, but are not limited to human lentiviruses such as HIV (in particular HIV-1 or HIV-2), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), Caprine Arthritis Encephalitis Virus (CAEV) and the VISNA virus.
• the genetic material also comprises at least one target polynucleotide, in particular one target polynucleotide, for which variants are desired.
• the particles containing this target polynucleotide are said "parentaf, since the target polynucleotide has not undergone yet the mechanisms of genetic variability resulting from both the retrotranscription and particle production following the implementation of the method of the invention.
• the determination of the "parental" feature of said lentiviral particles or qualification of prepared lentiviral particles as “parental” is performed or decided prior to implementing the method of the invention i.e., it refers to the characteristics, especially to the nucleotide sequence, enabling to recognize the target polynucleotide as the original polynucleotide for which a diversity is sought.
• the target polynucleotide found in the genetic material is heterologous to the viral particle i.e., the target polynucleotide is not identical to any gene or coding sequence found in the retrovirus or lentivirus from which the viral particles of the invention is derived.
• the target polynucleotide is not of lentiviral or viral origin, and in particular has a bacterial, mammalian (especially human) or synthetic (non natural) origin.
• the target polynucleotide may correspond to an entire genomic region (for example a gene with its regulatory sequences), to a gene (with introns, if any), a coding sequence (without introns), a non-coding sequence (such as a promoter) or any nucleotide sequence, either natural, synthetic or hybrid, either as a DNA, RNA or cDNA.
• the target polynucleotide can lead to the synthesis of a functional RNA, or can be functional by itself (as, for instance, being target of binding by a protein) or encodes a protein.
• the target polynucleotide encodes a protein that is chosen in the group consisting of (a) a structural protein, (b) a functional protein, such as pro-drug activation proteins, signal transduction proteins or receptor binding proteins, (c) a protein with enzymatic activity, and in particular enzyme with kinase activity such as deoxycytidine kinase, and (d) a secreted protein, in particular interferons such as interferon ⁇ .
• target polynucleotide for directed evolution using this system is not restricted to protein-encoding polynucleotide and can also be constituted by DNA sequences (as recognition sites for protein binding) or sequences that lead to transcription of RNA sequences of interest.
• regulatory expression elements may be operatively linked to said target polynucleotide.
• the regulatory expression element comprises or is a "promoter” i.e., a regulatory region of DNA generally located upstream of the target polynucleotide (in the 5' region of the sense strand) that generally promotes gene transcription.
• promoter i.e., a regulatory region of DNA generally located upstream of the target polynucleotide (in the 5' region of the sense strand) that generally promotes gene transcription.
• it is a specific nucleotide sequence in DNA, flanking the start of the target polynucleotide which instructs an RNA polymerase where to start transcribing RNA.
• any promoter able to promote the expression of the target polynucleotide in a cell in particular a mammalian cell (e.g. a human cell) is included within the scope of the present invention.
• promoter is advantageously an exogenous (non- viral) promoter.
• exogenous non-viral promoter can include, for instance, elongation factor 1 -a (EF1 -a), CMV, SV40, beta globin and PGK.
• the genetic material also comprises a selectable marker gene, optionally under the control of a secondary regulatory expression element.
• selectable marker gene refers to a gene coding for a protein allowing selection of the cell transduced with the lentiviral particle of the invention.
• the gene may code for, but is not limited to, a protein conferring resistance to an antibiotic (e.g. puromycin), a surface protein such as CD69, or a protein encoding a fluorescent marker.
• the optional secondary regulatory expression element is defined as above for the regulatory expression element linked to the target polynucleotide.
• the two regulatory expression elements for examples two promoters
• the two regulatory expression elements be different, although it is clear to one skilled in the art that other methods of expression of the second gene could be also used, as the use of internal ribosome entry sequences (IRES).
• Promoters which may be used advantageously in accordance with the present invention include for example, EF1 -oc, CMV, SV40, beta globin and/or PGK.
• the genetic material of the invention comprises, in addition to regulatory elements necessary for the transfer, transcription and optionally expression of said genetic material into the genome of a cell, at least one target polynucleotide (in particular one target polynucleotide and/or in particular a heterologous target polynucleotide) under the control of a first promoter (in particular exogenous), at least a second exogenous promoter driving the expression of a selectable marker gene, at least one selectable marker gene (e.g. puromycin- resistance gene).
• a first promoter in particular exogenous
• a second exogenous promoter driving the expression of a selectable marker gene
• at least one selectable marker gene e.g. puromycin- resistance gene
• the genetic material, of the replication-defective lentiviral particles contemplated by the present invention comprises a 5' LTR, a first promoter driving the expression of a target polynucleotide, a selectable marker gene, a second promoter driving the expression of the selectable marker gene, and a 3' LTR, all of which are defined hereinabove.
• Examples of such genetic material are illustrated in Figures 3A to 3D. It is clear to a person skilled in the art, that the promoter sequences mentioned above can be replaced by other promoters, according to the desired strength of each promoter for the expression of each corresponding target polynucleotide or selectable marker gene. An inducible promoter can also be used, if desired. However it must be understood that the invention is not limited to the use of one target polynucleotide and a selectable marker gene and/or promoters, and less or more such entities can be used.
• the target polynucleotide is inserted in such a way that the target polynucleotide is operatively associated with the first promoter (this is not restrictive though, and the polynucleotide target of the evolution experiment could also be constituted by the second target polynucleotide).
• the target polynucleotide may be inserted between the first and the second promoters of the genetic material, in particular downstream the first promoter and upstream the second promoter.
• such target polynucleotide may code for deoxycytidine kinase (dCK).
• genetic material also called vector
• plasmid SDY-PdCK encoding the dCK gene ( Figure 3D)
• XL10 cells deposited at the CNCM (Collection Nationale de Cultures de Microorganismes; Institut Pasteur; Paris, France) under accession number 1-3991 on May 14, 2008 (XL10-SDY-PdCK), in the name of Institut Pasteur, 25-28 rue du Dondel Roux, 75724 Paris Cedex 15.
• the lentiviral particles used are said replication-defective i.e., once the lentiviral particle has entered into a cell, it cannot replicate alone to form new particles.
• the genes encoding the viral proteins necessary for the replication of the virus are not present or are defective, in the lentiviral particles.
• a defect may be due to a mutation and/or deletion of one or more viral structural and replication functions (e.g. the gag, pol and env genes) in the genetic material.
• the genetic material as described in detail above does not comprise the functional gag gene, the functional pol gene and/or the functional env genes of the lentivirus used as a basis to prepare the transfer vector carrying the genetic material.
• partial sequences of said genes may be present in the genetic material, such the ⁇ sequence (element overlapping the gag gene) or the cPPT-CTS region (a fragment derived from the pol gene).
• the genetic material does not comprise any of the coding genes normally comprised in the lentivirus or retrovirus genome.
• the replication-defective lentiviral particles of the invention are obtainable by a transcomplementation system (vector/packaging system).
• permissive cells such as 293T cells
• permissive cells are transfected in vitro with a plasmid containing the genetic material discussed above, and at least one plasmid providing, in trans, the gag, pol and env sequences encoding the polypeptides Gag, Pol and the envelope protein(s), or for a portion of these polypeptides sufficient to enable formation of retroviral or lentiviral particles.
• permissive cells are transfected with a first plasmid comprising the genetic material defined above (transfer vector), a second plasmid (envelope expression plasmid or pseudotyping env plasmid) comprising a gene encoding an envelope protein(s) and a third plasmid (encapsidation plasmid or packaging construct) expressing the Gag and Pol proteins.
• a first plasmid comprising the genetic material defined above (transfer vector)
• a second plasmid envelope expression plasmid or pseudotyping env plasmid
• a third plasmid encapsidation plasmid or packaging construct
• the transcomplementation step comprises or consists in transfecting in vitro a previously transduced producer cell with at least one plasmid providing, in trans, the gag, pol and env sequences encoding the polypeptides Gag, Pol and the envelope protein(s), or for a portion of these polypeptides sufficient to enable formation of retroviral or lentiviral particles.
• previously transduced producer cells are transfected with a first plasmid (envelope expression plasmid or pseudotyping env plasmid) comprising a gene encoding an envelope protein(s) and a second plasmid (encapsidation plasmid or packaging construct) expressing the Gag and Pol proteins.
• a first plasmid envelope expression plasmid or pseudotyping env plasmid
• a second plasmid encapsidation plasmid or packaging construct
• the replication-defective lentiviral particles of the invention may be pseudotyped with the envelope protein of the lentivirus used to prepare the lentiviral particles, or alternatively with a heterologous envelope protein that is chosen with respect to the cells to be targeted.
• these envelope proteins are amphotropic (wide host range).
• said lentiviral vector is pseudotyped with a VSV-G protein.
• the VSV-G glycoprotein may originate from different serotypes of vesiculoviruses.
• said lentiviral particles are pseudotyped with a protein chosen in the group consisting of the MuLV amphotropic envelope, the Mokola envelope, the EboZ envelope, the Ebola-Reston (EboR) envelope, the influenza-hemagglutinin (HA) envelope, the respiratory syncytial virus (RSV) F and G, the Venezuelan equine encephalitis, the Western equine encephalitis and and rabies virus envelope proteins.
• a protein chosen in the group consisting of the MuLV amphotropic envelope, the Mokola envelope, the EboZ envelope, the Ebola-Reston (EboR) envelope, the influenza-hemagglutinin (HA) envelope, the respiratory syncytial virus (RSV) F and G, the Venezuelan equine encephalitis, the Western equine encephalitis and and rabies virus envelope proteins.
• the parental replication-defective lentiviral particles as defined above are used for transducing producer cells.
• transducing refers to the process of introducing the genetic material as described herein into the cytoplasm of a cell, in particular producer cells, following the contact of this cell with the replication-defective lentiviral particles (either parental or the following generated particles such as F1 to Fn+1 ) with these cells.
• the envelope proteins of the replication-defective lentiviral particles are chosen according to the nature of the cells in which the genetic material has to be integrated.
• the genetic material is transported into the cell cytoplasm.
• the genetic material under the form of RNA molecule
• the genetic material is reverse transcribed, using the proteins found in the core of the particle, in double stranded DNA that is then imported into the nucleus and integrated into the genome of the cell.
• Producer cells that can be used in the present method are well known from the person skilled in the art.
• producer cells are of human origin, such as the Human Embryonic Kidney (HEK) 293T cells (ATCC CRL-1573). If other lentiviruses are used, the producer cells can be of mammalian origin in general, depending on the type of lentivirus used.
• the transduction of producer cells may be carried out in the presence of a mutagen, by exposing producer cells to the mutagen (for example during 48 hours) simultaneously to the transduction of replication-defective lentiviral particles.
• the mutagens used should preferentially be mild mutagens in order to limit the introduction of mutations that could be deleterious, if too frequent, for the sequences essential for the functionality of the lentiviral vector.
• mutagens that may be used are the 5-hydroxy-2'-desoxycytidine (5-OH-dC) (Loeb et al. 1999) or the KP-1212, administered as the prodrug KP-1461 (Anderson et al. 2004, Daifuku et al. 2003 and Harris et al. 2005) (see also "Dangerous Properties of Industrial Materials", 7th Ed., by N. Irving Sax and Richard J.
• Hela cells were seeded in 48 wells plates at 40,000 cells/well in the presence of 8 ⁇ of aphidicolin (Sigma). Cells were transduced with the replication-defective lentiviral particles as defined above, at a concentration ranging from 1 to 100 ng/ml, 24 hours after the aphidicolin block, which was replenished in the medium at the time of transduction;
• FL-DC dendritic cells
• step c producer cells that have been transduced with the replication- defective lentiviral particles (parental or F1 to Fn), are then transcomplemented with viral proteins necessary for the production of a new generation of replication-defective lentiviral particles (Fn to Fn+1 ).
• the host cell is lacking the proteins (both enzymatic and structural) enabling the production of new lentiviral particles (i.e., Gag, Pol and Env proteins).
• these proteins are provided by transfecting the transduced producer cells with at least one plasmid harbouring an env gene (preferably VSV-G) and gag and pol genes of a retrovirus or lentivirus.
• these proteins are provided transfecting the transduced producer cells with a first plasmid harbouring an env gene (preferably VSV-G) (such as the pHCMV-G plasmid) and with a second plasmid harbouring gag and pol genes of a retrovirus or lentivirus (such as the pCMVAR.2 plasmid).
• transfecting refers to the introduction of a nucleic acid into a cell, preferably by the intermediary of a plasmid, such as for example by calcium phosphate transfection or using cationic polymers or lipopolyamine (for example polyethyleneimine or PEI).
• a plasmid such as for example by calcium phosphate transfection or using cationic polymers or lipopolyamine (for example polyethyleneimine or PEI).
• new replication-defective lentiviral particles of the first filial generation of from the Fn to Fn+1 are synthesized and produced in the supernatant of the producer cell culture.
• these new replication-defective lentiviral particles are collected and separated from producer cells (step d).
• An example of process for collecting new replication-defective lentiviral particles includes filtration (for example with a 0.45 micron filter) and then concentration using ultrafiltration, such as centrifugal filtration (for example using Vivaspin from Sartorius Stedim biotech).
• steps b) to d) are repeated consecutively n times, wherein n is 1 or more (step e).
• steps b) to d) are repeated consecutively n times, wherein n is 1 or more (step e).
• steps b) to d) are repeated consecutively n times, wherein n is 1 or more (step e).
• steps b) to d) are repeated consecutively n times, wherein n is 1 or more (step e).
• the replication- defective lentiviral particles obtained after step b) to d) are from the first filial generation (F1 ). They are used to implement step e) to generate the second generation of replication-defective lentiviral particles (F2). These F2 particles are then used to implement step e) to generate the third generation of replication-defective lentiviral particles (F3), and so on to generate, after implementing n times the whole step e), the n+1 generation of replication-defective lentiviral particles (Fn+1 ).
• a population (set or mix) of replication-defective lentiviral particles of the n+1 filial generation is obtained, the particles containing indifferently the target polynucleotide or any nucleotide variant of this target polynucleotide. Therefore, the population of particles contains different nucleotide variants of the same target polynucleotide. Indeed, following each transduction step (b and e1 ), the RNA molecules contained in the replication-defective lentiviral particles is reverse transcribed in double strand DNA before integration into the genome.
• each retrotranscription step leads to one nucleotide error for 3x10 5 retrotranscribed nucleotides.
• the complexity of the variants of the target polynucleotide may be increased by the recombination carried out between the two RNA molecules contained in the same replication-defective lentiviral particles. These two mechanisms cause the production of variants of the target polynucleotide.
• target polynucleotide it is meant the polynucleotide inserted in the genetic material used to produce the parental replication-defective lentiviral particles (P) and whose nucleotide sequence is the target for the generation of mutation(s) after implementation of the method of the invention, giving rise to target polynucleotide variants.
• target polynucleotide variant refers to a nucleotide sequence similar to the sequence of the target polynucleotide i.e., having at least 80%, at least 90%, at least 95% or at least 99% similarity over the whole length with the sequence of the target polynucleotide.
• Target polynucleotide variants may also be defined by the fact they differ from the sequence of the target polynucleotide by one or more nucleotide substitutions (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10), deletion(s) and/or insertion(s).
• the variants may in some embodiments also be defined by the fact that the one or more nucleotide substitutions (as defined above) leading to amino acid, conservative, semi-conservative or non-conservative, substitution(s) in the resulting protein variants.
• the expression "protein variant' refers to a sequence similar to the sequence of the protein (the protein encoded by the target polynucleotide) i.e., having at least 80%, at least 90%, at least 95% or at least 99% similarity over the whole length with the sequence of the protein.
• Variants may also be defined by the fact that they differ from the sequence of the protein by one or more amino acid, conservative, semi-conservative or non-conservative, substitution(s) (e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10), deletion(s) and/or insertion(s).
• transduced producer cells may be optionally overtransduced with replication-defective lentiviral particles of the same generation as the ones used to transduce the producer cell lines, and obtained from separate cultures of the previous transcomplemented producer cell lines.
• step e) also comprises before step e2) overtransducing m consecutive times, wherein m is at least 1 , the transduced producer cells of step e1 ) with replication-defective lentiviral particles of the same immediately previous generation as the ones used in step e1 ), optionally in the presence of a mutagen.
• the conditions of overtransduction are the same as the conditions used in step e1 ) for the transduction step.
• overtransducing refers to the process of transduction, at least two consecutive times, of transduced producer cells (already transduced by replication-defective lentiviral particles of the same generation) with replication-defective lentiviral particles.
• the replication-defective lentiviral particles used for the m consecutive overtransductions (m is at least 1 ) have been collected from cultures separately transcomplemented (in space) from the producer cells which give rise to the Fn+1 cell generation, the latter being used for overtransduction.
• Each step e) of the method (repeated n times) is chosen independently from the previous and/or following steps e) of the method.
• the method comprises a first step e) without overtransduction, followed by a second step e) with overtransduction:
• step e1 transducing fresh producer cells with the immediately previous generation of replication- defective lentiviral particles (Fn-1 ), optionally in the presence of a mutagen, thereby obtaining the Fn filial cell generation; this step is similar to step e1 ) disclosed above.
• transcomplementing in parallel, several cultures of transduced producer cells of step e1 ) with viral proteins necessary for the production of a n generation of replication-defective lentiviral particles (Fn) and obtaining several cultures of producer cells producing a n generation of replication-defective lentiviral particles; "in parallel' means that identical cultures, but separated in space, are transcomplemented as explained before;
• step e4 transducing fresh producer cells with the replication-defective lentiviral particles (Fn) obtained from one of the cultures of step e2), optionally in the presence of a mutagen;
• step e5 overtransducing m consecutive times the transduced producer cells of step e4) with the replication-defective lentiviral particles obtained from the other several cultures of producer cells of the previous step e2) under the same conditions as step e4), optionally in the presence of a mutagen, wherein m is at least 1 ; in this step, "consecutive" has the same meaning as above.
• Replication-defective lentiviral particles obtained from the identical, but separated in space, cultures obtained in step e2) are used to transduce already transduced producer cells (overtransduction).
• the method of the invention may also optionally comprise after each time the producer cells have been transduced for the first time with replication-defective lentiviral particles (i.e, not after over-transduction), a step of selecting the producer cells having integrated in their genome the genetic material contained in the replication-defective lentiviral particles. This selection step may be carried out by any method enabling to determine the correct integration of the genetic material (PCR, detection of LTR).
• the selection is carried out by detecting the protein encoded by this gene (by ELISA or FACS) or by detecting the effect of this gene (for example resistance to antibiotics).
• the selectable marker gene is an antibiotic-resistance gene such as the puromycin-resistance gene. It is noteworthy that it may be useful, after each transduction step, to isolate transduced clones of producer cells for sequencing all or part of the heterologous target polynucleotide inserted in the genome of the producer cell of step (d) to analyse the genetic variability generated (variants of the target polynucleotide).
• the selectable marker gene is involved in antibiotic resistance
• cells that have not been transduced or that have not integrated in their genome the reverse transcription product of the genetic material will be removed, for example, by treatment with an antibiotic (e.g. puromycin).
• an antibiotic e.g. puromycin
• the cells containing the selectable marker gene can be stored at any cycle or after n times, providing a stock for restarting the experiment if accidental technical problems are encountered during the subsequent cycles.
• the present invention also discloses an in vitro method of generating a cell library expressing genetic diversity variants of a target polynucleotide, comprising:
• target ce/ refer to any cell whose the particular phenotype to study is known i.e., that the behaviour of the target cell regarding a particular function (enzymatic activity, molecule secretion, cell interaction, ...) is known.
• Target cells are used for highlighting the presence of a desired phenotype after transduction with the lentiviral vectors described in the present invention.
• Target cells might, or might not, be different from the "producer cells" described above.
• the target cell is a mammalian cell, and in particular a human cell, such as Hela TK- cells, cells lines deleted for SLP-76 or reporter HL1 16 cells.
• the producer cells used in the various steps of the method of the invention are all of the same type and are of the same type as the target cell.
• different producer cells are used during the procedure, according to the requirements of the specific experiment.
• these producer cells are of a different type with respect to the target cell.
• the replication-defective nature of the lentiviral vector allows a clonal analysis of the variants of the target polynucleotide, since the absence of any further viral replication, after the n th step of transduction, in the transduced target cells (step g of the method) results in the controlled delivery of only one target polynucleotide variant (optionally encoding a variant protein) in the target cell. Therefore, the cells of the library differ from each other by the fact they contain, integrated into their genome, the target polynucleotide or any variant of the same target polynucleotide, as a result of the variability introduced in the target polynucleotide following the transduction steps and optionally overtransduction steps.
• the present invention also relates to a method of screening a variant of a target polynucleotide associated with a cell phenotype comprising:
• step g) of this method being carried out on target cells having an original phenotype
• target cells having an original phenotype or “original target cells”, it is meant a target cell as defined above, in which a phenotype of interest is observed when said cells have not been transduced by the replication-defective lentiviral particles or before their transduction with replication-defective lentiviral particles.
• a "phenotype” is defined as any observable property of the target cell, which may be put in evidence directly (on the target cell itself) or indirectly (not on the target cell).
• a phenotype may correspond to a physical, biochemical or physiological trait of the target cell, observable on the cell or following interaction of the cell with its environment.
• the phenotype of interest is chosen according to the nature of the target polynucleotide inserted in the genetic material in step a) of the method to generate the cell library. Examples of phenotypes are disclosed below:
• secondary cells means that the phenotype of the transduced target cells is revealed on cells that are put in contact with the transduced target cell;
• “Final phenotype” is defined as the phenotype (as defined above) observed once the genetic material of the replication-defective lentiviral particles has been integrated in the genome of the target cell. Since the only genetic difference between the non-transduced target cells and the transduced target cells is the integration of the retrotranscribed product of the genetic material contained in the replication-defective lentiviral particles, the observed final phenotype results necessarily from the integration of the target polynucleotide (optionally, encoding a protein).
• Table 1 lists the difference of phenotype that can be observed and the means to detect this difference. Examples of mean of
• secondary cells in on the secondary cells e.g.
• Table 1 Examples of phenotype that can be observed on transduced target cells and examples of means to detect a difference in the phenotype after transduction; 3 : positive selection on a negative genetic background, b : negative selection on a positive genetic background, c : negative selection on a negative genetic background.
• the term "isolated” or “isolating” means, when referring to the phenotype of a target cell, that the target cell has a separate and discrete final phenotype with respect to the other target cells of the culture or library.
• the isolation of the target cells with the final phenotype can be performed by any method known to a person skilled in the art.
• target polynucleotide in its original or variant version
• different isolation or selection methods may be used to select the target cells that have incorporated in their genome the genetic material encoding the target polynucleotide variant which is associated with the desired phenotype.
• the isolation step of the present invention may occur by (i) positive selection on a negative genetic background, by (ii) negative selection on a negative genetic background, or by (iii) negative selection on a positive genetic background. If, however, the target polynucleotide is associated with the expression of or encodes a secreted protein, selection may occur by positive selection on secondary cells ( Figure 7).
• a target polynucleotide is considered to be associated with the expression of a protein, when modification in this target polynucleotide influences (e.g. inhibits, decreases or increases) the expression of this protein, though the coding sequence of this protein is not modified or altered. This can be the case, for example, of transcription or expression regulatory elements, such as promoter, enhancer, ...
• Positive selection on a negative genetic background can be performed by transduction of target cells (preferably human target cells) that do not exhibit the activity or do not express the desired molecule and selection for those target cells that have acquired the desired activity or express the desired molecule.
• the isolation step comprises isolating target cells having a desired activity or expressing a desired molecule, whereas the original target cells before transduction in step g) respectively lack the desired activity or do not express the desired molecule
• the transduction of target cells that do not exhibit the activity or do not express the desired molecule is performed. Selection for the transduced target cells that have incorporated in their genome the target polynucleotide and that do not express the activity or do not express the desired molecule may then occur. As such, the selection of target cells that comprise the selectable marker gene, but do not exhibit the desired phenotype is involved. Of note, the presence of the additional selectable marker gene (as resistance to an antibiotic, therefore not linked to the phenotype desired) is essential for this type of selection.
• the isolation step comprises isolating the target cells transduced as in step g) that (1 ) have stably integrated the reverse transcription product generated by the lentiviral vector and (2) have not acquired the desired activity or do not express a desired molecule, as instead would have been found if the transgene carried by the vector encoded a functional product.
• a variant of the screening based on negative selection is constituted by performing negative selection on a positive genetic background. By this means one can identify dominant negative mutants, which confer a negative phenotype to originally positive cells.
• the isolation step comprises isolating the target cells which, once put in contact with secondary cells, are able to activate or inhibit the activity of these secondary cells or the expression of molecules by these secondary cells (for example by activating the production of a fluorescence reporter molecule).
• a variant of the target polynucleotide is targeted.
• the supernatant from the clones isolated on the basis of the expression of the selectable marker is applied to secondary cells in order to assess the activation of a reporter gene in the secondary cells due to the action of the secreted molecule whose expression is associated with or which is encoded by the variant of the target polynucleotide.
• the variant is identified by any means enabling to distinguish it from the target polynucleotide, ideally through the phenotype of the target cell. If not possible, other approaches could be followed as, for example by restriction fragment length polymorphism (RFLP) or on arrays.
• RFLP restriction fragment length polymorphism
• the variation may also be identified on the encoded protein, for example by measuring the isoelectric point or measuring the size on polyacrylamide gel.
• the target polynucleotide is sequenced and the variation at the level of the nucleotide sequence and optionally at the level of the protein sequence is determined.
• the sequencing is advantageously carried out by amplifying (for example by PCR) the sequence of the target polynucleotide, since the sequences surrounding the target polynucleotide (in 5' and in 3') are perfectly known and thus primers for amplification may be easily designed (see for example Figure 3D).
• the present invention also relates to a cell library, preferably a mammalian cell library, in particular a human cell library, comprising cells having integrated in their genome a target polynucleotide or a nucleotide variant of said target polynucleotide, wherein said cell library is obtained following the transduction by a population of replication-defective lentiviral particles of a generation from F4 to F20, preferably F8 to F16, or of at least the 8 th , 10 th , 12 th or 15 th generation, these replication-defective lentiviral particles being obtained previously in the absence or presence of a mutagen.
• a cell library preferably a mammalian cell library, in particular a human cell library, comprising cells having integrated in their genome a target polynucleotide or a nucleotide variant of said target polynucleotide, wherein said cell library is obtained following the transduction by a population of replication-defective lentiviral particles of
• the term “genome” refers to any nucleic acid molecule that is stably present in the target cell, in particular whose presence into the cell is not dependent upon pressure selection.
• the term “genome” does not encompass plasmids.
• the term “genome” refers to nucleic acid molecules present in the cell nucleus (nuclear genome), by opposition to nucleic acid molecules present in the cytoplasm, and encompasses for example chromosomes.
• the term “genome” also includes nucleic acid molecules present in particular cell compartments, such as organelles, for example mitochondria (mitochondrial genome) or chloroplasts (chloroplast genome).
• All the cells or most of the cells in the library differ from each others in the sequence of the integrated target polynucleotide or variant of this same target polynucleotide, as a result of the variability introduced in the target polynucleotide following the transduction steps and optionally overtransduction steps.
• the replication-defective lentiviral particles used for the transduction of the target cells differ in the sequence of the target polynucleotide.
• replication-defective lentiviral particles of the at least 8 th generation means replication defective lentiviral particles (as defined above) obtained after implementing the method, of generating genetic diversity in the nucleotide sequence of a target polynucleotide, of the invention, wherein n is at least 7; in a particular embodiment, the method is implemented with at least m steps of overtransdcution as defined above.
• the cells of the library have integrated in their genome a retrotranscribed target polynucleotide or retrotranscribed target polynucleotide having a sequence different from the original sequence (target polynucleotide variant).
• All cells of the library are characterized by the fact that they have integrated into their genome the genetic material as defined above i.e., at least the regulatory elements necessary for integration (for example the LTR), the target polynucleotide or the target polynucleotide variant (optionally encoding a protein or its variant) under the control of regulatory expression elements.
• the genetic material also contains a selectable marker gene, this selectable marker gene may be used to isolate the cells of the library that have effectively integrated the genetic material into their genome.
• Such a cell library is the library 293T-F8dCK deposited at that CNCM under accession number 1-3992 on May 14, 2008, in the name of Institut Pasteur, 25-28 rue du Dondel Roux, 75724 Paris Cedex 15.
• This cell library is a population of HEK-293 T cells corresponding to the 8 th generation obtained by the method of the invention, and containing integrated in its genome the dCK gene or variant thereof, as the target polynucleotide.
• This cell population may be cultivated in Dubelcco-Modified Eagle's Medium supplemented with 10% fetal calf serum, 100U/ml penicillin, 0.1 mg/ml streptomycin and 0.6 ⁇ g ml puromycin, at a temperature of 37°C, in 5% C0 2 .
• the present invention also relates to a nucleic acid comprising or consisting in an U3 region of a HIV-1 LTR mutated in positions 24, 82, 108, 160, 164, 183, 198, 251 , 291 , 333, 421 and 447 with respect to the U3 region of a wild type LTR of HIV-1 virus.
• the mutated U3 region is characterized by the mutations 24A>C, 82A>G, 108G>A, 160OA, 164T>G, 183A>G, 198A>C, 251 G>A, 291 G>A, 333T>C, 421 >G and 4470T with respect to the U3 region of a wild type LTR of HIV-1 virus, in particular with respect to SEQ ID NO:5.
• the mutated U3 region has the sequence as defined in SEQ ID NO:6.
• the U3 region may be obtained following the implementation of the claimed method in human cells, synthesized chemically or obtained by directed mutagenesis of a wild type U3 region.
• This mutated U3 region confers an advantage, in human cells, in particular in HEK 293T cells, for the expression of a target polynucleotide from the LTR or from the genetic material as described herein, and is used for this optimized expression.
• This mutated U3 region is hypothesized to favour ectopic expression in human cells of the target polynucleotide by expressing higher content of genomic RNA (RNA transcribed from the genetic material which has been inserted into the cell genome) than the wild type U3 region.
• the invention also concerns a nucleic acid comprising or consisting in a LTR, in particular a HIV-1 LTR, in which its U3 region is mutated in the positions described above or has the mutations as defined above or has a sequence as defined in SEQ ID NO:6.
• the invention is also directed to the genetic material as defined herein, a lentiviral vector as defined herein comprising this genetic material or a particle as defined herein comprising this genetic material, in which the U3 region of its LTR 5' and/or LTR 3', preferably the LTR3', is mutated in the positions described above or has the mutations as defined above or has a sequence as defined in SEQ ID NO:6.
• the invention also concerns any lentiviral vector, i.e., a vector derived from a lentivirus, in particular a HIV vector or a HIV-1 vector, in which the U3 region of its LTR 5' and/or LTR 3', preferably the LTR3', is mutated in the positions described above or has the mutations as defined above or has a sequence as defined in SEQ ID NO:6.
• a cell, a cell culture or a cell library comprising inserted in its genome a genetic material in which the U3 region of its LTR 5' and/or LTR3' is mutated in the positions described above or has the mutations as defined above or has a sequence as defined in SEQ ID NO:6.
• the invention also relates to the use, in particular in vitro or in cell culture, of replication-defective lentiviral particles comprising a genetic material encapsidated in said particles, wherein said genetic material comprises the regulatory elements necessary for the transfer, transcription and optionally expression of said genetic material in the genome of a cell host and at least one target polynucleotide, for generating diversity in the nucleotide sequence of said target polynucleotide and/or for generating a cell library expressing genetic diversity variants of said target polynucleotide. All definitions and features disclosed above apply to the use of the replication-defective lentiviral particles.
• the main advantage of the method of the present invention is that a library of variants of the target polynucleotide is generated directed in the biological vector (lentiviral particles) that is used to deliver efficiently and in a controlled manner the target polynucleotide to the target cell (in particular the human cell).
• lentiviral particles biological vector
• a crucial feature of the invention is the use of replication-defective lentiviral particles that can be brought back to controlled replication competence, by a transcomplementation system. The replication-defective nature of these particles allows clonal screening for the desired phenotype in the target cells.
• a further advantage of the method contemplated by the present invention is the use of retroviral recombination, through a procedure that favours it, named overtransduction, to amplify the genetic complexity of the library.
• an advantage of retroviral or lentiviral transduction of cells (with particles) with respect to transfection (directly with nucleic acids) is that, by modulating the multiplicity of transduction steps, one can limit the introduction of the target polynucleotide (original sequence or a sequence variant) to not more than one copy per target cell. This is a prerequisite to perform clonal analysis based on the phenotype conferred to the target cell by a single target polynucleotide.
• the use of replication-defective (retroviral or lentiviral) particles for delivering the target polynucleotide allows to rescue, in the transduced target cell presenting the desired phenotype, the target polynucleotide variant responsible for that phenotype. If a virus were used (as in reference Das et al.) the virus would continue to replicate during selection for the desired phenotype and reinfect the target cells in culture with other variants of the target polynucleotide generated in the meantime. This would hamper clonal expansion. Selection for the phenotype conferred by a single target polynucleotide variant would not be possible as neither would be the identification of the target polynucleotide variant responsible for that given phenotype.
• replication-defective particles for delivering the target polynucleotide does not induce any cytopathic effect.
• replication-defective particles for delivering the target polynucleotide allows to pseudotype the viral particles with an amphotropic envelope (as that of the Vescicular Stomatitis Virus; VSV), allowing transduction of a wide range of cells.
• the library can be used to transduce (and therefore screen for the desired phenotype) also primary and non-replicative (or non-dividing) cells.
• the method of the present invention allows direct screening of the desired phenotype in target cells (eukaryotic, mammalian or human cells). More physiologically relevant observations with respect to screening in simpler systems, such as binding in vitro of purified proteins or bacterial genetic screening can thus be obtained with the method of the present invention.
• target cells eukaryotic, mammalian or human cells. More physiologically relevant observations with respect to screening in simpler systems, such as binding in vitro of purified proteins or bacterial genetic screening can thus be obtained with the method of the present invention.
• another advantage of the present invention is the fact that target polynucleotide variants that are toxic for the target cells will automatically be excluded during the evolution procedure. In fact, their isolation will likely not occur since, during the procedure of generation of genetic diversity, the target polynucleotide is repeatedly introduced in the cells (through transduction in producer cells) where it is expressed. If toxic, these cells would be eliminated from the culture.
• the methods of the invention lie on the genetic diversity generated by the two following mechanisms.
• the Fn+1 viral particles (generated by transfecting the cells of the Fn generation) are collected and used to transduce producer cells (for instance HEK 293T cells) that are then selected for their resistance conferred by the resistance marker gene.
• producer cells for instance HEK 293T cells
• the procedure followed is the one outlined in Figures 5A and 5B.
• the target polynucleotide will evolve by accumulating modifications and recombination events during reverse transcription.
• the complexity of the library i.e., the number of variants of the target polynucleotide
• the complexity of the library i.e., the number of variants of the target polynucleotide
• reverse transcription is also responsible for the generation of other important genomic rearrangements, among which the most frequent is recombination.
• Another immediate advantage of a particular embodiment of the method of the present invention is the overtransduction step, described in the present application. Indeed, to ensure the presence of multiple copies of genetic material in the same producer cell, the method of the present invention is based on multiple successive overtransduction steps ( Figures 5A and 5B). As such, once a cell population is established (e.g. the Fn population), cells are transcomplemented with helper plasmids to generate the next generation of replication- defective particles (Fn+1 ) which will be then used to transduce freshly producer cells and optionally to select the transduced producer cells (for example with antibiotics such as the puromycin to select Fn+1 resistant cell population).
• a cell population e.g. the Fn population
• helper plasmids to generate the next generation of replication- defective particles (Fn+1 ) which will be then used to transduce freshly producer cells and optionally to select the transduced producer cells (for example with antibiotics such as the puromycin to select Fn+1 resistant cell population).
• the Fn cell population may be maintained in culture and used to produce further Fn+1 replication-defective particles which may then be used to transduce previously-transduced Fn+1 cells (overtransduction step).
• Overtransduction can be reiterated, in principle without limits in the number of times.
• lentiviral particles i.e., lentiviral particles harbouring two different molecules of genetic material
• recombination brings about several point modifications in the same genetic background in a single infectious cycle, increasing the repertoire of genetic combinations explored.
• Copy choice occurs during reverse transcription as a consequence of template switching by the reverse transcriptase between the two copies of genomic RNA (herein defined as genetic material) present in the viral particle, a process known to a person skilled in the art as "copy choice”. Copy choice occurs at rates of approximately 10 "4 events per nucleotide and per infectious cycle when considering similar sequences.
• recombination is expected to increase the complexity of variants at two levels.
• the parental replication-defective lentiviral particles (P) are generated, in step a) of the method, in permissive cells using, besides the encapsidation plasmid and the envelope expression plasmid, a transfer vector containing the at least two related genes as target polynucleotides or at least two transfer vectors, each containing one related gene as target polynucleotide.
• RNAs obtained following the transcription of the integrated genetic material possess the same dimerization signal, dimerization of the at least two related RNAs (each from the related target polynucleotide) occurs on a stochastic basis, leading to the generation of heterozygous replication-defective lentiviral particles that carry one copy of each related target polynucleotide. Recombination between these two related target polynucleotides can then occur during reverse transcription.
• Another application of the invention is that overtransduction by replication-defective lentiviral particles containing in their genetic material selectable marker gene(s) different from the one carried for the transduction step (and also possibly containing in its genetic material a different version of the target polynucleotide from the one carried for the transduction step) can be followed by double selection to selectively isolate the population of cells that have been both transduced and then overtransduced by the two types of replication-defective lentiviral particles. This would allow the evolution of two or more target polynucleotides simultaneously.
• the evolving target polynucleotides could be genes belonging to the same family of genes, or can be related genes (as for instance desoxyribonucleoside kinases, as indicated in the specific examples provided herein). Interestingly, in this case, recombination can also lead to the generation of chimeric genes between these target polynucleotides.
• Plasmids The SDY-PdCK plasmid was constructed in the laboratory and the map is provided in Figure 3D.
• pCMVAR8.2 (Naldini L, Blomer U., Gallay P., Ory D., Mulligan R., Gage F.H., Verma I.M. and Trono D. (1996) Science, 272, 263-267) is a plasmid encoding HIV-1 Gag, Pol, and accessory proteins, and pHCMV-G (Yee J.K., Miyanohara A., LaPorte P., Bouic K., Burns J.C. and Friedmann T.
• HEK 293T cells are grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin and streptomycin (Invitrogen), and maintained at 37°C with 5% C0 2 . Nomenclature of Cell Lines and Viral Generations
• V-FXdC Each viral generation is named V-FXdC, where "V” stands for virus (replication-defective lentiviral particles), “FX” for the generation ("FP” stands for parental viral generation; “F3” stands for third viral generation) and “dC” indicates that the target polynucleotide for the evolution is the dCK gene. Nucleotide sequence of the dCK gene and its corresponding protein sequence are disclosed in SEQ ID NO:1 and SEQ ID NO:2 respectively. For cells, the same procedure is followed replacing V by C, for cells. Therefore, as an example, C-F3dC cells generate V-F4dC particles that, once used to transduce HEK 293T cells, generate the C-F4dC cell line. Generation of the Parental Cell Line
• Concentrated supernatant is then used to transduce 5x10 6 fresh HEK 293T cells in a final volume of 5 ml in the presence of 2.5 ⁇ of polybrene.
• Cells are first incubated 5 hours at 37°C in 60 mm Petri dishes non-treated for cell culture, and then transferred to 10 cm cell culture dishes and further incubated for 20 hours at 37°C. The cells are then detached by trypsin-EDTA treatment and re-seeded in the presence of 0.6 ⁇ g ml puromycin. Once the puromycin-resistant population selected (C-FPdC) the cells are used to continue the experiment and be re-infected. Generation of the Clear Cell Lines by Transduction
• C-FPdC cells are then transfected with pCMVAR8.2, and pHCMV-G at a weight ratio of 2:1 , and treated as described in the previous paragraph up to the transduction of fresh HEK 293T cells and the establishment of the next cell generation (C-F1 dC) through puromycin selection. The procedure is then repeated generating the C-F2dC cell line, and the subsequent generations up to the F5 cell line (C-F5dC).
• C-F5dC cells are transfected as described above for the generation of particles (V- F6dC).
• V-F6dC particles treated as described above, are used to generate C-F6dC cells.
• C-F5dC cells are maintained in culture and used to generate new V-F6dC particles that are used for transduction, once established by puromycin selection, the C-F6dC cell line. This is what we define the overtransduction procedure as shown in Figure 5B. This generates the C- F6dC2 cell line. Over-transduction with V-F6dC is then done on C-F6dC2 cells, generating the C-F6dC3 cell line.
• the procedure is repeated four (4) times to generate the C-F6dC5 cell line.
• cells are cultured in the presence of puromycin.
• the C-F6dC5 cell line is used to generate the new generation of particles (V- F7dC).
• the whole procedure can be repeated, to generate the following generations of particles.
• Viral preparation from V-F8dC has been diluted 1 :10,000 and used for transduction of 3.5 x 10 6 HEK 293T cells as described above. Five hours post-transduction, 1 .5 ml of transduced cells are diluted to a final volume of 25 ml and seeded in 96-well plates (100 ⁇ /well). Twenty-four hours later, puromycin is added to a final concentration of 0.6 ⁇ g ml. Single puromycin-resistant clones are then isolated, expanded, and genomic DNA extracted for sequencing of the dCK gene.
• the cell pellet from a 10 cm plate is resuspended in 250 ⁇ of lysis reagent (Viagen Biotech, Los Angeles, CA, USA) with 1 ug/ml of proteinase K and incubated overnight at 55°C.
• the cell lysate is then incubated for 1 hour at 86°C and 1 ⁇ used as template for PCR amplification with primers PGK-CR and EF1 -CR that anneal on the PGK and EF1 -oc promoters, respectively, as indicated in Figure 3D.
• PCR products are then sequenced.
• dCK deoxycytidine kinase
• FIG. 6A shows sixteen (16) independent clones from the F8 generation.
• Figure 6A shows, at the top, the fourteen (14) positions that have been found modified in at least one of the sixteen (16) clones analyzed, and their nucleotide position.
• Concerning the modification pattern of the clones analyzed it is to be noted that when the same position is found in different clones, the same nucleotide substitution was involved. Certain positions indicate G to A transitions (1 14, 136, 232, 237, 379, 478, 563, 739, 751 , 763), A to G transitions (106 and 1 16), C to A transversions (745), and deletion of one nucleotide (173).
• the positions indicate G to A transitions (237, 478, 51 1 , 521 and 739), A to G transitions (106 and 504), C to T transitions (625 and 661 ), a T to C transition (472), a C to A transversion (745), a T to A transversion (251 ) and a deletion of 27 nucleotides (939-419 del).
• Modification at positions 251 , 393-419del, 472, 504, 51 1 , 521 , 625, 656-657 and 661 are new modified positions with respect to the modifications identified at the F8 generation.
• Deoxynucleoside kinases are clinically relevant since they activate prodrugs used in antiviral and anticancer therapy, as for ganciclovir a compound used in about 10% of cancer gene therapy in clinical trials (Wiewrodt R., Amin K., Kiefer M., Jovanovic V.P., Kapoor V., et al. (2003) Cancer Gene Ther., 10: 353-64).
• ganciclovir a compound used in about 10% of cancer gene therapy in clinical trials.
• the 80 individual cellular clones at the F17 generation obtained after transduction of 293T cells at a low multiplicity of infection (and therefore carrying a single variant of the target polynucleotide) have been isolated.
• the 80 clones have been tested for the kinetic of cell death induced after their exposal to different concentrations of the two anti-cancer drugs, AraC and Gemcitabine, which are cytidine analogues (the natural substrate of dCK) as follows.
• a relative rate of cell death has been calculated for each clone, with respect to the values observed in the plate where no drug was included. These values will then be compared to those observed for control clones.
• These controls have been constituted by three puro-resistant independent cell clones isolated after transduction with vectors carrying the wild type dCK enzyme. Three independent controls have been tested to consider that a potential variability might be present even with the same dCK sequence, due to possible epigenetic effects. The expression of the dCK gene could indeed be modulated by the position of integration of the proviral DNA within the target cell genome.
• a dCK variant (referred herein as G7, squares in Figure 8) displayed a clear tendency to induce cell death at concentrations of the prodrug (AraC) for which cell death was much lower than the wt sequence (triangle in Figure 8).
• the G17 variant has been demonstrated to contain a G to A transition at position 521 , leading to the G174E variation at the amino acid level.
• TM mutant described by Sabini et al. (see Table 2 above), for which no effect on the efficiency of phosphorilation of AraC was observed in vitro, no significant difference was displayed with respect to the wt dCK enzyme.
• a dCK variant of interest has been identified (mutant G12), disclosing a G to A transition at position 51 1 (E171 K at the amino acid level).
• the G12 variant induced cell death in a higher proportion of cells when tested at 10 nM Gemcitabine (the lowest concentration, among those tested, at which a differential effect was observed between G12 and the wt dCK) on twice as much cells than the dCK gene (ratio of cell survival in G12 cells / dCK cells: 0.50 ⁇ 0.1 1 , average of 4 independent experiments). In the TM mutant (Table 2 above), this ratio was 0.89 ⁇ 0.08 (4 experiments).
• the Messa 10K cells are uterine sarcoma cells defective for the expression of the dCK gene (Jordheim LP, Galmarini CM, and Dumontet C, 2006). Due to the lack of a functional dCK protein, these cells display a high degree of resistance to the deoxycytidine analogues (Gemcitabine IC50 :1 1 uM) and are an ideal background in which to test the ability of the library of dCK variants to increase the sensitivity to Gemcitabine. Indeed, the phenotype produced by the introduction of a single copy of a dCK variant will be the result of this variant and would not be covered by the activity of several copies of wild type dCK.
• the expression of the dCK gene variants could be modulated by the position of integration of the proviral DNA within the target cell genome, through epigenetic effects. These effects constitute a major problem for screening procedures, since they can lead to identification of false positive or to ignore interesting mutants.
• a strategy for the screening on Messa 10K cells was set up, aiming at averaging the epigenetic effects possibly influencing the final phenotype by analysing a population of target cells containing the same dCK variant inserted in different sites of the genome.
• viral vectors or particles have been rescued from a subset of HEK293T isolated clones by transfection with transcomplementation plasmids and used, separately, to transduce Messa 10K cells, at a M.O.I. ⁇ 1 . Each transduction gave rise to 20- 200 cellular clones, bearing the same dCK variant inserted in different parts of the genome.
• the four rounds of screening on Messa 10K cells confirmed that the claimed method (retrovolution procedure) enables the isolation of a cell line (293T-G12-F16) producing viral vectors that, once inserted in human cells, confer a phenotype of increased sensitivity to Gemcitabine.
• the stability and reproducibility of the phenotype was verified by submitting the G12-F17 vectors or particles (rescued from G12-F16 cells) to a further passage: the viral vectors F17 were used to transduce fresh 293T cells to produce the 293T-G12 F17 cells, a new generation of vectors was produced by transfection with transcomplementation plasmids (G12- F18) and these were used to transduce Messal OK cells at low M.O.I.
• SLP-76 an adaptor protein involved in signalling in response to T cell receptor activation
• the goal of targeting an adaptor protein is to provide an example of how our directed evolution system is adapted for studying the functionality of this important type of proteins.
• activation of signalling pathways depends on several enzymes such as protein kinases and phosphatases, phospholipases, GTPases, but also on scaffolds and adaptors. These proteins are responsible for the recruitment of effectors in supra-molecular complexes, where the ordered activation of the various binding partners is regulated.
• the activation of production and expression at the surface of the cell of the CD69 molecule can also be followed.
• the goal is to find variants that are affected in at least one of the three readouts (NFAT- promoter-driven luc expression, IL-2-promoter-driven luc expression, and CD69 expression).
• Differential effects on the three readouts would be an interesting result opening on the identification of mutations of SLP-76 that lead to a differential phenotype. This would be important for dissecting how the same adaptor can control differential activation pathways.
• Modification of this gene is expected to provide an example of the selection for negative phenotypes that our system allows (see Figure 7) due to the possibility of first selecting cells containing a target polynucleotide through the selection for puromycin positive cells, followed by selection for a negative phenotype conferred by the target polynucleotide variant.
• SLP-76-/- cells having a negative genetic background but allowing the correct readout are required.
• SLP-76-/- cells were already available (Jurkat J14 cells SLP-76-/-). These cells allow screening based on the expression of CD69.
• the IL-2-luc Jurkat cell lines and NFAT luc Jurkat cell lines are transduced with the SLP-76 particles from the library obtained by the disclosed method of the invention.
• target cells having the Puror phenotype resistance to puromycin
• the induction of the expression of the luciferase is measured for each clone and compared to that observed in the presence of the wild type SLP-76 gene.
• EXAMPLE 3 GENETIC VARIABILITY GENERATED IN THE GENE ENCODING THE INTERFERON LAMBDA IFN ⁇ exhibits several common features with type I IFNs since they share a signalling pathway involving the transcription factor ISGF3, driving the expression of a common set of responsive genes (Dumoutier L., Lejeune D., Hor S., Fickenscher H., Renauld J.C. (2003) Biochem. J. 370: 391 -6; Kotenko S.V., Gallagher G., Baurin V.V., Lewis-Antes A., Shen M., et al. (2003) Nat.
• IFN ⁇ is able of establishing an antiviral state in sensitive cell lines and it possesses an antiproliferative activity (Meager A., Visvalingam K., Dilger P., Bryan D., Wadhwa M. (2005) Cytokine, 31 : 109-18). IFN ⁇ is of potential interest with respect to type I IFNs, due to the cell specific expression of their receptor in contrast to the ubiquitous presence of receptors for type I IFNs.
• the method of the present invention is implemented to obtain variants with higher specific activities for the induction of ISGF3, especially on cells expressing low levels of IFN ⁇ receptor.
• Two kinds of effects of variants of IFN ⁇ are expected:
• - IFN ⁇ variant having an increased efficiency that fully mimic type I IFNs; this kind of variant is useful in clinic to reduce the side effects of IFN therapy since the spectrum of responsive organs is restricted by the expression of the IFN ⁇ receptor.
• This is interesting, for instance, for the treatment of HCV infections where a direct antiviral effect on hepatocytes (a cell type that expresses the IFN receptor) is wanted.
• - IFN ⁇ variant behaving qualitatively like an ordinary type I IFN i.e., exhibiting antiviral, antiproliferative and Th1 driver bioactivity.
• IFNs .are able to suppress hepatitis C viral replication in HuH-7 cells Zhu H., Butera M., Nelson D.R., Liu C. (2005) Virol. J., 2: 80. It is conceivable that IFN variants could be of use in clinic to treat such a viral infection and the spectrum of responsive organs being restricted by the expression of IFNLR1 , the treatment would be less likely to cause the myelosuppression and other side effects typically associated with type I IFN therapy (Brassard D.L., Grace M.J., Bordens R.W. (2002) J. Leukoc. Biol., 71 : 565-81 ).
• the method of the invention has now been implemented on the IFN ⁇ target polynucleotide, and the fourth generation of viral particles has been reached.
• One possible problem that could have occur is the possible modification of the properties of the 293T cells once transduced by a vector that encodes for the IFN ⁇ , since IFN ⁇ has an antiviral activity.
• this problem has not been encountered for leading the successive steps of transfection and transduction, indicating that this is not the case.
• Table 3 luciferase activity observed in HL-1 16 cells exposed to the culture supernatant from 17 independent clones isolated after transduction of 293T cells
• the method of the invention may be applied to genes of different nature (enzyme, adaptators, secreted proteins) to produce a library of cells expressing a diversity of variants of a target polynucleotide; and

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